Effect of caspase inhibitors on the post-thaw motility, and integrity of acrosome and plasma membrane of cryopreserved equine spermatozoa

The present study was designed to test the hypothesis that addition of anticaspase cocktails (inhibiting caspases and thus blocking apoptosis) to the extenders increases the post-thaw viability of equine spermatozoa. The addition of caspase inhibitors failed to improve the acrosome and plasma membrane integrity of spermatozoa, suggesting that in equine sperm cryopreservation protocols, the addition of these caspase inhibitors to cryopreservation medium may not be beneficial in protecting the sperm from the stress of cryopreservation.     

Efficacy of the anticaspase agent zVAD-fmk on post-thaw viability of canine spermatozoa

Cryopreservation protocols for gametes are constantly improved with the aim of increasing the post-thaw viability of gametes. It is becoming clear that stress, resulting from cryopreservation, reduces cell numbers by apoptosis. Apoptosis, or programmed cell death, is a gene-activated event that occurs as a normal consequence of development and as a result of cellular stress. Apoptosis is mediated by the family of cysteine-dependent asparate-specific proteases (caspases). The present study was designed to test the hypothesis that addition of an anticaspase (zVAD-fmk) that has inhibitory properties against caspases and apoptosis to semen extenders and to the thaw medium would increase post-thaw viability of canine spermatozoa. Extenders were added in a two-step process. A dose of 100 microM caspase inhibitor was used. Four groups (n=6 for each) were composed based on the presence or absence of the caspase inhibitor: Group I (control), no caspase inhibitor in the extender or the thaw medium; Group II, caspase inhibitor in the thaw medium; Group III, caspase inhibitor in Extender II; and Group IV, caspase inhibitor in both Extender II and the thaw medium. Post-thaw motility, plasma membrane integrity, and acrosome status were investigated. The addition of caspase inhibitor to Extender II or to the thaw medium failed to improve the parameters that were studied. The results suggest that this caspase inhibitor may not be beneficial to the post-thaw motility of canine spermatozoa if used at this concentration.     

Cell death in pancreatitis: caspases protect from necrotizing pancreatitis

Mechanisms of cell death in pancreatitis remain unknown. Parenchymal necrosis is a major complication of pancreatitis; also, the severity of experimental pancreatitis correlates directly with necrosis and inversely with apoptosis. Thus, shifting death responses from necrosis to apoptosis may have a therapeutic value. To determine cell death pathways in pancreatitis and the possibility of necrosis/apoptosis switch, we utilized the differences between the rat model of cerulein pancreatitis, with relatively high apoptosis and low necrosis, and the mouse model, with little apoptosis and high necrosis. We found that caspases were greatly activated during cerulein pancreatitis in the rat but not mouse. Endogenous caspase inhibitor X-linked inhibitor of apoptosis protein (XIAP) underwent complete degradation in the rat but remained intact in the mouse model. Furthermore, XIAP inhibition with embelin triggered caspase activation in the mouse model, implicating XIAP in caspase blockade in pancreatitis. Caspase inhibitors decreased apoptosis and markedly stimulated necrosis in the rat model, worsening pancreatitis parameters. Conversely, caspase induction with embelin stimulated apoptosis and decreased necrosis in mouse model. Thus, caspases not only mediate apoptosis but also protect from necrosis in pancreatitis. One protective mechanism is through degradation of receptor-interacting protein (RIP), a key mediator of "programmed" necrosis. We found that RIP was cleaved (i.e. inactivated) in the rat but not the mouse model. Caspase inhibition restored RIP levels; conversely, caspase induction with embelin triggered RIP cleavage. Our results indicate key roles for caspases, XIAP, and RIP in the regulation of cell death in pancreatitis. Manipulating these signals to change the pattern of death responses presents a therapeutic strategy for treatment of pancreatitis.     

Study of apoptosis-related markers in ram spermatozoa

Certain features of capacitated or frozen-thawed spermatozoa have been considered to be an apoptosis-like phenomenon, and, it has been suggested that the presence of apoptotic sperm in seminal doses could be one of the reasons for poor fertility. The objective of this study was to determine whether phosphatidylserine (PS) translocation, caspase activity and DNA fragmentation, which are considered to be apoptotic markers in somatic cells, occur in ram sperm. Fresh ejaculates and sperm samples in different physiological state (cold-shocked, in vitro capacitated and acrosome-reacted (AR)) were compared. Simultaneous staining with 6-carboxifluorescein diacetate (6-CFDA) and Annexin V-Cy3.18 (AnnV) revealed four different sperm subpopulations in ejaculates. The main subpopulation was composed of viable cells without PS exposure (CFDA+/AnnV-). A total of 40.8% of sperm showed inverted PS, with two levels of alteration: CFDA+/AnnV+ in midpiece ("type I AnnV+"), and in acrosome and midpiece ("type II AnnV+"). The fewest subpopulation contained non-viable cells showing Annexin labelling in the entire cell (CFDA-/AnnV+). Labeling of caspases-3 and -7 by immunocytochemistry revealing different sperm subtypes depending on their localization in apical, equatorial, post-acrosomal regions and tail. The results obtained by western-blot showed, for the first time to our knowledge, that caspase-like proteins are present in fresh ram semen as both inactive and active forms. The proportion of sperm with fragmented DNA [terminal transferase-mediated dUDP nick end-labeling (TUNEL)-positive] were found rarely (2.7+/-0.5%) in all fresh ejaculates involved in this study. The analysis of total activity of both caspases by a fluorometric method showed a decrease in vitro capacitated and acrosome-reacted samples as well as in cryoinjured samples. However, the percentage of TUNEL-positive sperm demonstrating DNA fragmentation was significantly increased after in vitro induced capacitation and acrosome reaction, as well as after cold-shock although this augment was not significant. PS exposure is not totally dependent on caspases in ram spermatozoa as the addition of a caspase inhibitor prevented the increase in PS inversion due to incubation in capacitating conditions but not to the ionophore-induced acrosome reaction or cold-shock.     

Tyrosine phosphoproteome of hamster spermatozoa: Role of glycerol‐3‐phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine‐phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol‐3‐phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation.     

Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro

In previous reports from this laboratory, we identified the presence of a novel alpha-D-mannosidase on the surface of rat, mouse, hamster, and human spermatozoa [J Cell Biol 1989; 109:1257-1267 and Biol Reprod 1990; 42:843-858]. Since it has been suggested that mannosyl residues on the egg zona pellucida may be important for sperm-egg binding, studies were undertaken to examine the potential role of the sperm alpha-D-mannosidase during fertilization. Incubation of mouse spermatozoa in the presence of increasing concentrations of the inhibitory sugars, alpha-methyl mannoside, alpha-methyl glucoside, D-mannose, or D-mannitol, resulted in a dose-dependent decrease in the number of spermatozoa bound per egg without a deleterious effect on sperm motility or on the sperm acrosome, and a dose-dependent inhibition of the sperm mannosidase activity. Galactose, however had no effect on sperm-egg binding or on sperm mannosidase activity. Two nucleotide sugars (UDP-GlcNAc and UDP-gal) were also tested and shown to reduce sperm-egg binding but with only a minimal effect on sperm mannosidase activity. In additional studies, spermatozoa incubated in the presence of a mannose-containing oligosaccharide exhibited a dramatic reduction in sperm-egg binding that correlated with a similar inhibition of sperm mannosidase activity. The oligosaccharide substrate did not affect sperm motility or the sperm acrosome. These studies suggest that the sperm alpha-D-mannosidase may play an important role during fertilization.     

In vitro studies of the golden hamster sperm acrosome reaction: completion on the zona pellucida and induction by homologous soluble zonae pellucidae

We have studied the occurrence of the golden hamster sperm acrosome reaction (AR) in vitro during interaction with the oocyte investments: the cumulus cell matrix and the zona pellucida. Hamster sperm were capacitated in a defined medium that does not induce the AR. These spermatozoa were allowed to interact with the ovum vestments, the events of which were recorded using high-speed videomicrography. Frame-by-frame analysis revealed that sperm did not complete the AR in the cumulus cell matrix, but did so on the zona pellucida. Furthermore, a higher percentage of sperm completed the AR on the zona pellucida of cumulus-invested than on cumulus-free eggs. We also investigated the effect of solubilized hamster and mouse zonae pellucidae on the hamster sperm AR. Addition of solubilized hamster zonae to capacitated sperm elicited the AR within 15 min. Solubilized mouse zonae were significantly less effective, indicating that the zona-induced AR in hamster sperm may be species specific. These results suggest that the hamster zona pellucida is an inducer of the AR in the intact or soluble form, and that the majority of spermatozoa traverse the cumulus cell matrix without completing the AR in our in vitro system.     

Hamster sperm capacitation: role of pyruvate dehydrogenase A and dihydrolipoamide dehydrogenase

Recently, we demonstrated that pyruvate dehydrogenase A2 (PDHA2) is tyrosine phosphorylated in capacitated hamster spermatozoa. In this report, using bromopyruvate (BP), an inhibitor of PDHA, we demonstrated that hamster sperm hyperactivation was blocked regardless of whether PDHA was inhibited prior to or after the onset of hyperactivation, but the acrosome reaction was blocked only if PDHA was inhibited prior to the onset of the acrosome reaction. Further, inhibition of PDHA activity did not inhibit capacitation-associated protein tyrosine phosphorylation observed in hamster spermatozoa. It is demonstrated that the essentiality of PDHA for sperm capacitation is probably dependent on its ability to generate effectors of capacitation such as reactive oxygen species (ROS) and cAMP, which are significantly decreased in the presence of BP. MICA (5-methoxyindole-2-carboxylic acid, a specific inhibitor of dihydrolipoamide dehydrogenase [DLD]), another component of the pyruvate dehydrogenase complex (PDHc), also significantly inhibited ROS generation and cAMP levels thus implying that these enzymes of the PDHc are required for ROS and cAMP generation. Furthermore, dibutryl cyclic adenosine monophosphate could significantly reverse the inhibition of hyperactivation observed in the presence of BP and inhibition of acrosome reaction observed in the presence of BP or MICA. The calcium ionophore, A23187, could also significantly reverse the inhibitory effect of BP and MICA on sperm acrosome reaction. These results establish that PDHA is required for hamster sperm hyperactivation and acrosome reaction, and DLD is required for hamster acrosome reaction. This study also provides evidence that ROS, cAMP, and calcium are involved downstream to PDHA.     

Isolation and proteomic characterization of the mouse sperm acrosomal matrix

A critical step during fertilization is the sperm acrosome reaction in which the acrosome releases its contents allowing the spermatozoa to penetrate the egg investments. The sperm acrosomal contents are composed of both soluble material and an insoluble material called the acrosomal matrix (AM). The AM is thought to provide a stable structure from which associated proteins are differentially released during fertilization. Because of its important role during fertilization, efforts have been put toward isolating the AM for biochemical study and to date AM have been isolated from hamster, guinea pig, and bull spermatozoa. However, attempts to isolate AM from mouse spermatozoa, the species in which fertilization is well-studied, have been unsuccessful possibly because of the small size of the mouse sperm acrosome and/or its fusiform shape. Herein we describe a procedure for the isolation of the AM from caput and cauda mouse epididymal spermatozoa. We further carried out a proteomic analysis of the isolated AM from both sperm populations and identified 501 new proteins previously not detected by proteomics in mouse spermatozoa. A comparison of the AM proteome from caput and cauda spermatozoa showed that the AM undergoes maturational changes during epididymal transit similar to other sperm domains. Together, our studies suggest the AM to be a dynamic and functional structure carrying out a variety of biological processes as implied by the presence of a diverse group of proteins including proteases, chaperones, hydrolases, transporters, enzyme modulators, transferases, cytoskeletal proteins, and others.     

Tumor necrosis factor induces distinct patterns of caspase activation in WEHI-164 cells associated with apoptosis or necrosis depending on cell cycle stage.

TNF is unusual among the death receptor ligands in being able to induce either apoptotic or necrotic cell death. We have observed that in WEHI 164 fibrosarcoma, cells the mode of TNF-induced cell death is dependent on the stage of the cell cycle. Cells arrested in G(0)/G(1) undergo necrosis, while those progressing through the cell cycle undergo apoptosis. TNF induces caspase activity in both settings, and the broad spectrum caspase inhibitor zVAD-fmk inhibits this activity and blocks both TNF-induced apoptosis and necrosis. Inhibition of oxygen radical accumulation does not block cytotoxicity. The presence and activation of specific caspases were examined by Western blotting. The procaspase-8a isoform was down-regulated in proliferating cells. Procaspases-8b and -7 were cleaved during TNF-induced apoptosis but not necrosis. Thus, a different pattern of caspase expression and activation occurs dependent on the cell cycle and which may determine the mode of cell death.     

Characterization of testicular mouse glucosamine 6-phosphate deaminase (GNPDA).

Mammalian glucosamine 6-phosphate deaminase (GNPDA) was first detected in hamster spermatozoa. To further elucidate its role, we have cloned mouse GNPDA and produced a polyclonal rabbit anti-GNPDA antibody. This antibody recognized a 33 kDa protein in soluble extracts from mouse brain, liver, kidney, muscle, ovary, testis and sperm. Immunofluorescent analysis of the localization of GNPDA in male reproductive tissue revealed its presence in spermatids and in spermatozoa. In spermatids, GNPDA localized close to the developing acrosome vesicle and in spermatozoa close to the acrosomal region. Following the induction of the acrosome reaction, GNPDA fluorescence in spermatozoa was either reduced or GNPDA was absent. These data suggest that GNPDA might play a role in the acrosome reaction.     

THE IMPORTANCE OF HYDROLYTIC ENZYMES TO AN EXOCYTOTIC EVENT, THE MAMMALIAN SPERM ACROSOME REACTION

The mammalian sperm acrosome reaction is a unique form of exocytosis, which includes the loss of the involved membranes. Other laboratories have suggested the involvement of hydrolytic enzymes in somatic cell exocytosis and membrane fusion, and in the invertebrate sperm acrosome reaction, but there is no general agreement on such an involvement. Although reference was made to such work in this review, the focus of the review was on the evidence (summarized below) that supports or fails to support the importance of certain hydrolytic enzymes to the mammalian sperm acrosome reaction. Because the events of capacitation, the prerequisite for the mammalian acrosome reaction, and of the acrosome reaction itself are not fully understood or identified, it is not yet always possible to determine whether the role of a particular enzyme is in a very late step of capacitation or part of the acrosome reaction. (1) The results of studies utilizing inhibitors of trypsin-like enzymes suggest that such an enzyme has a role in the membrane events of the golden hamster sperm acrosome reaction. The enzyme involved may be acrosin, but it is possible that some as yet unidentified trypsin-like enzyme on the sperm surface may play a role in addition to or instead of acrosin. Results obtained by others with guinea pig, ram and mouse spermatozoa suggest that a trypsin-like enzyme is not involved in the membrane events of the acrosome reaction, but only in the loss of acrosomal matrix. Such results, which conflict with those of the hamster study, may have been due to species differences or the presence of fusion-promoting phospholipase-A or lipids contaminating the incubation media components, and in one case to the possibly damaging effects of the high level of calcium ionophore used. The role of the trypsin-like enzyme in the membrane events of the hamster sperm acrosome reaction may be to activate a putative prophospholipase and/or to hydrolyse an outer acrosomal or plasma membrane protein, thus promoting fusion. A possible role of the enzyme in the vesiculation step rather than the fusion step of the acrosome reaction cannot be ruled out at present. (2) Experiments utilizing inhibitors of phospholipase-A₂, as well as the fusogenic lysophospholipid and cis-unsaturated fatty acid hydrolysis products that would result from such enzyme activity, suggests that a sperm phospholipase-A₂ is involved in the golden hamster sperm acrosome reaction. Inhibitor and LPC addition studies in guinea pig spermatozoa have led others to the same conclusion. The fact that partially purified serum albumin is important in so many capacitation media may be explained by its contamination with phospholipase-A and/or phospholipids. Serum albumin may also play a role, at least in part, by its removal of inhibitory products released by the action of phospholipase-A₂ in the membrane. The demonstration of phospholipase-A₂ activity associated with the acrosome reaction vesicles and/or the soluble component of the acrosome of hamster spermatozoa, and the fact that exogenous phospholipase A₂ can stimulate acrosome reactions in hamster and guinea pig spermatozoa, also support a role for the sperm enzyme. The actual site or the sites of the enzyme in the sperm head are not yet known. The enzyme may be on the plasma membrane as well as, or instead of, in the acrosomal membranes or matrix. A substrate for the phospholipase may be phosphatidylcholine produced by phospholipid methylation. It is possible that more than one type of ‘fusogen’ is released by phospholipase activity (LPC and/or cis-unsaturated fatty acids, which have different roles in membrane fusion and/or vesiculation. In addition to acting as a potential ‘fusogen’, arachidonic acid released by sperm phospholipase-A₂ probably serves as precursor for cyclo-oxygenase or lipoxygenase pathway metabolites, such as prostaglandins and HETES, which might also play a role in the acrosome reaction. Although much evidence points to a role for phospholipase-A₂, phospholipase-C found in spermatozoa could also have a role in the acrosome reaction, perhaps by stimulating events leading to calcium gating, as suggested for this enzyme in somatic secretory cells. (3) A Mg²⁺-ATPase H⁺-pump is present in the acrosome of the golden hamster spermatozoon. Inhibition of this pump by certain inhibitors of ATPases (but not by those that only inhibit mitochondrial function) leads to an acrosome reaction only in capacitated spermatozoa and only in the presence of external K⁺. The enzyme is also inhibited by low levels of calcium, and such inhibition, combined with increased outer membrane permeability to H⁺ and K⁺, and possibly plasma membrane permeability to H⁺ (perhaps by the formation of channels), may be part of capacitation and/or the acrosome reaction. The pH of the hamster sperm acrosome has been shown to become more alkaline during capacitation, and such a change may result in the activation of hydrolytic enzymes in the acrosome or perhaps in a change in membrane permeability to Ca²⁺. A similar Mg²⁺-ATPase has not been found in isolated boar sperm head membranes. However, that conflicting result could have been due to the use of noncapacitated boar spermatozoa for the preparation of the membranes or to protease modification of the boar sperm enzyme during assay. (4) Inhibition of Na⁺, K⁺-ATPase inhibits the acrosome reaction of golden hamster spermatozoa, and the activity of this enzyme increases relatively early during capacitation. A late influx of K⁺ is important for the acrosome reaction. However, this late influx may not be due to Na⁺, K⁺-ATPase, but instead may be due to a K⁺ permeability increase (possibly via newly formed channels) in the membranes during capacitation. It is suggested in this review that Na⁺, K⁺-ATPase has a role early in capacitation rather than directly in the acrosome reaction (although such a role cannot yet be completely ruled out). One possible role for the enzyme in capacitation might be to stimulate glycolysis (which appears to be essential for capacitation and/or the acrosome reaction of hamster and mouse spermatozoa). The function of the influx of K⁺ just before the acrosome reaction is probably to stimulate, directly or indirectly, the H⁺-efflux required for the increase in intraacrosomal pH occurring during capacitation. Direct stimulation of the acrosome reaction by a change in membrane potential resulting directly from K⁺-influx is not a likely explanation for the hamster results. However, the importance of an earlier membrane potential change, due to increased Na⁺, K⁺-ATPase during capacitation, and/or of later membrane potential changes resulting from the pH change, cannot be ruled out. Although K⁺ is required for the hamster acrosome reaction, other workers have reported that K+ inhibits guinea pig sperm capacitation. However, the experimental procedures used in the guinea pig sperm studies raise some questions about the interpretation of those inhibition results. (5) Ca²⁺-influx is known to be required for the acrosome reaction. Others have suggested that increased Ca²⁺-influx due to inhibition or stimulation of sperm membrane calcium transport ATPases are involved in the acrosome reaction. There is as yet no direct or indirect biochemical evidence that inhibition or stimulation of such enzymatic activity is involved in the acrosome reaction, and further studies are needed on those questions. (6) I suggest that the hydrolytic enzymes important to the hamster sperm acrosome reaction will also prove important for the acrosome reaction of all other eutherian mammals.     

9-AAP, a fluorescent beta-adrenergic antagonist, enters the hamster sperm acrosome in a manner inconsistent with binding to beta-adrenergic receptors

We have previously shown that the hamster sperm acrosome reaction, an event essential for fertilization, is stimulated by beta-adrenergic agonists and inhibited by beta-adrenergic antagonists. In the present report, we describe attempts to use (+/-)-9-aminoacridylpropranolol (9-AAP), a fluorescent derivative of a potent beta-adrenergic antagonist, to microscopically detect beta-adrenergic receptors on cauda epididymal hamster spermatozoa in vitro. 9-AAP binding to washed hamster spermatozoa was localized primarily in the acrosomal region, but we were unable to consistently displace the 9-AAP with the biologically active (-)-stereoisomers of several beta-adrenertic agonists and antagonists. Such displacement is necessary in order to separate binding to receptors from "nonspecific binding." Thus we did not detect sperm beta-adrenertic receptors by this method. Failure to detect the receptors with 9-AAP may be due to their presence in numbers too low for detection by this compound or to the masking of the receptors in these uncapacitated sperm. However, we could displace 9-AAP with either 5.0 mM NH4Cl or 1.2 microgram/ml nigericin, compounds capable of discharging pH gradients across cell membranes. These compounds have also been previously reported to displace the fluorescent portion of 9-AAP, 9-aminoacridine from the acrosome by such mechanism. The present results suggest that 9-AAP fluorescence does not always represent binding to beta-adrenergic receptors or "nonspecific binding," but may also represent the concentration of 9-AAP in acidic compartments within a cell.     

Mannose-binding molecules of rat spermatozoa and sperm-egg interaction

We have previously reported the occurrence and partial characterisation of an alpha-D-mannosidase activity on plasma membranes of rat, mouse, hamster and human spermatozoa. A soluble isoform of the rat sperm surface mannosidase was purified and polyclonal antibody raised. Since several reports have suggested that mannosyl residues on the rat, mouse and human zona pellucida may be involved in sperm-zona binding, studies were undertaken to examine the receptor-like role of mannose-binding molecules on rat spermatozoa. Sprague-Dawley rats (25-30-days old) were superovulated and eggs collected from the oviduct were treated with 0.3% hyaluronidase to remove the cumulus cells. Spermatozoa, collected from the cauda epididymis were capacitated for 5 h at 37 degrees C in 5% CO2 in air. The sperm-zona binding assay was performed in the presence of increasing concentrations of several sugars as well as preimmune and immune (anti-mannosidase or anti-mannose binding protein) IgG. Data from these studies show that: (1) significantly fewer sperm bound per egg in the presence of competitive inhibitors of mannosidase; (2) among the sugars examined, D-mannose was the most potent inhibitor causing 70% reduction in the number of sperm bound per egg; (3) anti-mannosidase or anti-mannose binding protein (but not preimmune) IgG showed a dose-dependent reduction in the number of sperm bound per egg; (4) anti-mannosidase IgG (but not anti-mannose binding protein IgG) showed a dose-dependent inhibition of sperm surface mannosidase activity; (5) the competitive inhibitors of mannosidase or the immune IgG had no effect on sperm motility or the sperm acrosome reaction. These result suggest that mannose-binding molecule(s) such as alpha-D-mannosidase or mannose-binding protein on the spermatozoa may recognise mannosyl residues on zona pellucida, and play a receptor-like role in sperm-egg interaction in the rat.     

Appearance of an intra-acrosomal antigen during the terminal step of spermiogenesis in the rat

Summary In a survey of sperm antigens in the rat, a new intra-acrosomal antigen was found using a monoclonal antibody MC41 raised against rat epididymal spermatozoa. The MC41 was immunoglobulin G₁ and recognized spermatozoa from rat, mouse and hamster. Indirect immunofluorescence with MC41 specifically stained the crescent region of the anterior acrosome of the sperm head. Immuno-gold electron microscopy demonstrated that the antigen was localized within the acrosomal matrix. Immunoblot study showed that MC41 recognized a band of approximately 165000 dalton in the extract of rat sperm from the cauda epididymidis. Immunohistochemistry with MC41 demonstrated that the antigen was first detected in approximately step-2 spermatids, and distributed over the entire cytoplasmic region of spermatids from step 2 to early step 19. The head region became strongly stained in late step-19 spermatids and then in mature spermatozoa. Distinct immunostaining was not found in the developing acrosome of spermatids throughout spermiogenesis. These results suggest that the MC41 antigen is a unique intra-acrosomal antigen which is accumulated into the acrosome during the terminal step of spermiogenesis.     

Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria

In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation of truncated Bid; moreover, no mitochondrial release of cytochrome c is observed. Bcl-2 overexpression slows down the onset of necrotic cell death. In the case of apoptosis, active caspases are released to the culture supernatant, coinciding with the release of lactate dehydrogenase. Following necrosis, mainly unprocessed forms of caspases are released. Both TNF-induced necrosis and necrosis induced by anti-Fas in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone are prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, while Fas-induced apoptosis is not affected.     

Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

Proteases for cell suicide: functions and regulation of caspases.

Caspases are a large family of evolutionarily conserved proteases found from Caenorhabditis elegans to humans. Although the first caspase was identified as a processing enzyme for interleukin-1beta, genetic and biochemical data have converged to reveal that many caspases are key mediators of apoptosis, the intrinsic cell suicide program essential for development and tissue homeostasis. Each caspase is a cysteine aspartase; it employs a nucleophilic cysteine in its active site to cleave aspartic acid peptide bonds within proteins. Caspases are synthesized as inactive precursors termed procaspases; proteolytic processing of procaspase generates the tetrameric active caspase enzyme, composed of two repeating heterotypic subunits. Based on kinetic data, substrate specificity, and procaspase structure, caspases have been conceptually divided into initiators and effectors. Initiator caspases activate effector caspases in response to specific cell death signals, and effector caspases cleave various cellular proteins to trigger apoptosis. Adapter protein-mediated oligomerization of procaspases is now recognized as a universal mechanism of initiator caspase activation and underlies the control of both cell surface death receptor and mitochondrial cytochrome c-Apaf-1 apoptosis pathways. Caspase substrates have bene identified that induce each of the classic features of apoptosis, including membrane blebbing, cell body shrinkage, and DNA fragmentation. Mice deficient for caspase genes have highlighted tissue- and signal-specific pathways for apoptosis and demonstrated an independent function for caspase-1 and -11 in cytokine processing. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits.     

Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm

Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G alpha q/11 and the beta 1 isoform of phospholipase C (PLC beta 1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Kd = 26 nM, Bmax = 30 pmol/mg) and low affinity (Kd = 1.6 microM, Bmax = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome- reacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 microM IP3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca(2+)-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca2+ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of G alpha q/11, PLC beta 1 and a functional IP3R in the anterior acrosomal region of mammalian sperm, as well as thapsigargin''s induction of the acrosome reaction, implicate IP3-gated calcium release in the mammalian acrosome reaction.     

Caspases Mediate 6-Hydroxydopamine-Induced Apoptosis but Not Necrosis in PC12 Cells

Abstract: The neurotoxin 6-hydroxydopamine (6-OHDA) induces apoptosis in the rat phaeochromocytoma cell line PC12. 6-OHDA-induced apoptosis is morphologically indistinguishable from serum deprivation-induced apoptosis. Exposure of PC12 cells to a low concentration of 6-OHDA (25 µ M ) results in apoptosis, whereas an increased concentration (50 µ M ) results in a mixture of apoptosis and necrosis. We investigated the involvement of caspases in the apoptotic death of PC12 cells induced by 6-OHDA, using a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), and compared this with serum deprivation-induced apoptosis, which is known to involve caspases. We show that zVAD-fmk (100 µ M ) completely prevented the apoptotic morphology of chromatin condensation induced by exposure to either 6-OHDA (25 and 50 µ M ) or serum deprivation. Furthermore, cell lysates from 6-OHDA-treated cultures showed cleavage of a fluorogenic substrate for caspase-3-like proteases (caspase-2, 3, and 7), acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin, and this was inhibited by zVAD-fmk. However, although zVAD-fmk restored total cell viability to serum-deprived cells or cells exposed to 25 µ M 6-OHDA, the inhibitor did not restore viability to cells exposed to 50 µ M 6-OHDA. These data show the involvement of a caspase-3-like protease in 6-OHDA-induced apoptosis and that caspase inhibition is sufficient to rescue PC12 cells from the apoptotic but not the necrotic component of 6-OHDA neurotoxicity.